Exposure to high-frequency electromagnetic field triggers rapid uptake of large nanosphere clusters by pheochromocytoma cells.

BACKGROUND: Effects of man-made electromagnetic fields (EMF) on living organisms potentially include transient and permanent changes in cell behaviour, physiology and morphology. At present, these EMF-induced effects are poorly defined, yet their understanding may provide important insights into consequences of uncontrolled (e.g., environmental) as well as intentional (e.g., therapeutic or diagnostic) exposure of biota to EMFs. In this work, for the first time, we study mechanisms by which a high frequency (18 GHz) EMF radiation affects the physiology of membrane transport in pheochromocytoma PC 12, a convenient model system for neurotoxicological and membrane transport studies. METHODS AND RESULTS: Suspensions of the PC 12 cells were subjected to three consecutive cycles of 30s EMF treatment with a specific absorption rate (SAR) of 1.17 kW kg-1, with cells cooled between exposures to reduce bulk dielectric heating. The EMF exposure resulted in a transient increase in membrane permeability for 9 min in up to 90 % of the treated cells, as demonstrated by rapid internalisation of silica nanospheres (diameter d ≈ 23.5 nm) and their clusters (d ≈ 63 nm). In contrast, the PC 12 cells that received an equivalent bulk heat treatment behaved similar to the untreated controls, showing lack to minimal nanosphere uptake of approximately 1-2 %. Morphology and growth of the EMF treated cells were not altered, indicating that the PC 12 cells were able to remain viable after the EMF exposure. The metabolic activity of EMF treated PC 12 cells was similar to that of the heat treated and control samples, with no difference in the total protein concentration and lactate dehydrogenase (LDH) release between these groups. CONCLUSION: These results provide new insights into the mechanisms of EMF-induced biological activity in mammalian cells, suggesting a possible use of EMFs to facilitate efficient transport of biomolecules, dyes and tracers, and genetic material across cell membrane in drug delivery and gene therapy, where permanent permeabilisation or cell death is undesirable.

An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.

Vimentin is a cytoskeletal intermediate filament protein that is expressed in mesenchymal cells and cancer cells during the epithelial-mesenchymal transition. The goal of this study was to identify vimentin-targeting small molecules by using the Tocriscreen library of 1120 biochemically active compounds. We monitored vimentin filament reorganization and bundling in adrenal carcinoma SW13 vimentin-positive (SW13-vim+) cells via indirect immunofluorescence. The screen identified 18 pharmacologically diverse hits that included 2 statins-simvastatin and mevastatin. Simvastatin induced vimentin reorganization within 15-30 min and significant perinuclear bundling within 60 min (IC50 = 6.7 nM). Early filament reorganization coincided with increased vimentin solubility. Mevastatin produced similar effects at >1 µM, whereas the structurally related pravastatin and lovastatin did not affect vimentin. In vitro vimentin filament assembly assays revealed a direct targeting mechanism, as determined biochemically and by electron microscopy. In SW13-vim+ cells, simvastatin, but not pravastatin, reduced total cell numbers (IC50 = 48.1 nM) and promoted apoptosis after 24 h. In contrast, SW13-vim- cell viability was unaffected by simvastatin, unless vimentin was ectopically expressed. Simvastatin similarly targeted vimentin filaments and induced cell death in MDA-MB-231 (vim+), but lacked effect in MCF7 (vim-) breast cancer cells. In conclusion, this study identified vimentin as a direct molecular target that mediates simvastatin-induced cell death in 2 different cancer cell lines.-Trogden, K. P., Battaglia, R. A., Kabiraj, P., Madden, V. J., Herrmann, H., Snider, N. T. An image-based small-molecule screen identifies vimentin as a pharmacologically relevant target of simvastatin in cancer cells.

Ultrastructure of pheochromocytoma: undescribed morphologic features.

We examined samples of human pheochromocytoma from 11 patients aged 30-70 years including one case of malignant pheochromocytoma with a view to identifying previously unreported ultrastructural details.We identified two types of nuclear inclusions consisting of irregularly shaped singular or multiple granulofibrillar formations with a typical concentric halo, on the one hand, and accumulations of egg-shaped structures consisting of granules and microfilaments, on the other. In some of the tumor cells, membrane-covered inclusions containing parallel laminar elements arranged in a paracrystalline, periodic fashion, or mega-mitrochondriae characterized by increased electrodensity of their matrix, and fibrillary material in the spaces between the cristae were present. A frequent finding consisted of typical ciliary formations, while rough/smooth tubular aggregates of different size occurred less frequently. Finally, we were able to demonstrate the uptake of norepinephrine by smooth muscle fibers in the periphery of arterial vessels as evidenced by linear accumulations of membrane-covered granules separating bands of contractile smooth muscle components in the peripheral layers of arterial vessels close to norepinephrine producing neoplastic cells.These findings represent ultrastructural features that contribute to further elucidating the ultrastructural characteristics of the human pheochromocytoma.

Ultrastructural findings in adrenal cortical adenomas clinically mimicking pheochromocytoma: a comparison with other adrenal tumors and tissue preparation techniques.

Adrenal cortical tumors clinically mimicking pheochromocytomas are extremely rare, with 14 cases in the literature. The authors describe 2 patients with adrenal cortical adenoma (ACA) and catecholamine elevations. The impact of tissue preparation methods on electron microscopy (EM) images was assessed in ACA mimicking pheochromocytoma, pheochromocytoma, and ACA lacking pheochromocytoma-like symptoms. Ten adrenal cortical tumors were examined using EM after a variety of tissue preparation techniques, including fixation with glutaraldehyde, formalin for varying lengths of time followed by glutaraldehyde, and/or formalin followed by paraffin embedding. Electron micrographs were assessed for image quality and the presence of dense secretory granules and eccentric, norepinephrine (NE)-type granules. Images created from tissue fixed in glutaraldehyde and/or formalin and embedded in resin were of good quality, while those derived from paraffin-embedded specimens were poor with disrupted cellular architecture. When pheochromocytoma was fixed in glutaraldehyde for 24 h or in formalin for 8 days, eccentric granules were identified. These granules were absent when tissue was fixed in formalin for 20 days or was obtained from a paraffin block. ACA without pheochromocytoma-like symptoms and ACA mimicking pheochromocytoma both had noneccentric dense-core granules on EM regardless of tissue preparation, and eccentric NE-type granules were absent. ACA is a rare cause of pheochromocytoma-like symptoms. These tumors lack eccentric, NE-type dense-core granules present in pheochromocytoma. Glutaraldehyde alone or formalin fixation followed by glutaraldehyde produces electron micrographs that may aid in the diagnosis of adrenal cortical tumors, whereas formalin-fixed, paraffin-embedded tissue results in images that are inadequate.

Adrenal imaging.

The differentiation of adrenal nodules is wide and varies from primary benign neoplasia, through hormone secreting lesions, to primary and secondary malignant masses. With the rapid development of cross-sectional imaging, incidental detection of adrenal nodules has become an everyday practice, leaving clinicians with the necessity of further investigation. In this article, we present the current possibilities of adrenal gland imaging and we propose a diagnostic schema for differential diagnosis of incidentaloma. Non-contrast enhanced computer tomography (CT) is a modality of choice used for the differential diagnosis of adrenal lesions. It allows the detection of 95% of adrenal masses, and the characterisation of most of them. Magnetic resonance imaging (MRI) is a good modality for cases in which CT examination cannot determine the character of an adrenal tumour. Nuclear medicine study with the use of Iodine-131 meta iodobenzylguanidine(MIBG) is helpful in the diagnosis of pheochromocytoma. Positron Emission Tomography-Computed Tomography (PET-CT) is considered a useful method in patients with a known malignancy history. Ultrasound has a low sensitivity for the detection of small lesions and is not capable of reliable characterisation of visualised masses. However, this technique plays an important role in the follow-up of non-hypersecreting adrenal lesions.

[Clinicomorphological aspects of adrenal pheochromocytoma diagnostics].

The removed pheochromocytomas of 25 patients aged from 25 to 72 years have been studied. Among them 5 tumors have had bilateral character, 8 pheochromocytomas have been diagnosed as malignant, 4 patients have had metastases. The average size of benign pheochromocytomas has been 4.0 cm, malignant - 4.5 cm. The majority of malignant tumors have had areas with diffusing growth patterns. The results of immunohistochemical study of benign and malignant pheochromocytomas have had small differences. The study of tumor ultrastructure hasn't found out their biological potential.

Cystic lymphangioma-like adenomatoid tumor of the adrenal gland: Case presentation and review of the literature.

Adenomatoid tumors (AT) are usually found in the genital tract of both sexes. They are very rarely located in extragenital sites, and are exceedingly rare in the adrenal. AT of the adrenal gland (AT-AG) are nonfunctioning, usually discovered incidentally and confused on imaging with other more common adrenal neoplasms. The overwhelming majority occur in males. Thirty-four cases have been reported so far, more often presenting grossly as solid tumors, rarely as solid with cystic areas, and 5 cases were almost entirely cystic. At histology they can be either circumscribed or locally infiltrative, and may pose diagnostic difficulties when the pathologist relies on morphology alone or is challenged on frozen section. On light microscopy the diagnosis may be very difficult if the tumor is rich in vacuolated cells, mimicking metastatic signet ring-cell adenocarcinoma. Immunophenotyping and/or electron microscopy are paramount in helping to ascertain their mesothelial lineage. Lymphangioma is the main histologic mimic of solid-cystic and cystic AT-AG, but lymphangioma is immunopositive for endothelial markers and negative for cytokeratins and mesothelial markers. Ultrastructural analysis has been performed in 10 published cases of AT-AG, in all of which the classical microvilli of coelomic type were always observed. In brief we report herein the sixth case of cystic lymphangioma-like AT, which was incidentally discovered during clinical follow-up in a 39-year-old man undergoing cancer staging and surveillance after surgery. The adrenal tumor was 5.5 cm in size and was fully investigated immunohistochemically and ultrastructurally. A complete review of the literature is also presented.

[Adenomatoid tumor of the right adrenal gland: a case report]. / Adenomatoidný tumor pravej nadoblicky: kazuistika.

Adenomatoid tumor of adrenal gland is a very rare primary tumor with favourable prognosis. The mesothelial origin of this tumor was confirmed by multiple studies of various authors. This origin was proven by immunohistochemical and ultrastructural examinations. In our case report, we present an interesting case of the adenomatoid tumor of the right adrenal gland in a 55-year-old woman. Our case is the second well-documented case of this tumor occurring in a female adult patient. We emphasize the presence of an intraluminal thread-like bridging strands, generally considered to be a characteristic histologic feature of this tumor, which have not yet been reported in literature in adenomatoid tumor located in adrenal glands.

Inhaled anesthetic enhancement of amyloid-beta oligomerization and cytotoxicity.

BACKGROUND: The majority of surgical patients receive inhaled anesthetics, principally small haloalkanes and haloethers. Long-term cognitive problems occur in the elderly subsequent to anesthesia and surgery, and previous surgery might also be a risk factor for neurodegenerative disorders like Alzheimer and Parkinson disease. The authors hypothesize that inhaled anesthetics contribute to these effects through a durable enhancement of peptide oligomerization. METHODS: Light scattering, filtration assays, electron microscopy, fluorescence spectroscopy and size-exclusion chromatography was used to characterize the concentration-dependent effects of halothane, isoflurane, propofol, and ethanol on amyloid beta peptide oligomerization. Pheochromocytoma cells were used to characterize cytotoxicity of amyloid oligomers with and without the above anesthetics. RESULTS: Halothane and isoflurane enhanced amyloid beta oligomerization rates and pheochromocytoma cytotoxicity in vitro through a preference for binding small oligomeric species. Ethanol and propofol inhibited oligomerization at low concentration but enhanced modestly at very high concentration. Neither ethanol nor propofol enhanced amyloid beta toxicity in pheochromocytoma cells. CONCLUSIONS: Inhaled anesthetics enhance oligomerization and cytotoxicity of Alzheimer disease-associated peptides. In addition to the possibility of a general mechanism for anesthetic neurotoxicity, these results call for further evaluation of the interaction between neurodegenerative disorders, dementia, and inhalational anesthesia.

Adrenal cortical adenoma with excess black pigment deposition, combined with myelolipoma and clinical Cushing's syndrome.

We report a case of a functional adenoma with excess black pigment deposition and myelolipoma in the same adrenal gland in a 58-year-old woman. The patient presented with gastrointestinal bleeding, and after being diagnosed with colonic diverticulosis, underwent a total colectomy. An abdominal computerized tomographic (CT) scan during her work-up showed a right adrenal mass consistent with myelolipoma. Postoperatively, the patient was diagnosed with Cushing's syndrome and underwent a right adrenalectomy. The adrenalectomy specimen consisted of a dark brown and golden-yellow adrenal adenoma, myelolipoma, and atrophic adrenal gland. Immunostains indicated that the dark brown adenoma component was responsible for the patient's hypercortisolism. Co-occurrence of a functional black adenoma and a well-developed myelolipoma has not been reported in the literature. We describe the significant findings of this case, together with a review of the literature.

An antigen retrieval method using an alkaline solution allows immunoelectron microscopic identification of secretory granules in conventional epoxy-embedded tissue sections.

Immunoelectron microscopy using chromogranin A-specific antibodies has been proposed as an efficient technique for identification of secretory granules (SGs) in tumor cells with evidence of apparent neuroendocrine differentiation. Using an antigen retrieval (AR) method, we succeeded in immunolabeling SGs with antibodies in ultrathin sections of routinely processed epoxy-embedded blocks of tissue. Samples of an insulinoma were fixed in 2% glutaraldehyde, postfixed in 1% OsO(4), and embedded in epoxy resin. Ultrathin sections were immunostained with chromogranin A-specific antibodies and gold-conjugated second antibodies. There was no significant labeling in the absence of AR. Neither etching with sodium metaperiodate nor microwave irradiation of ultrathin sections in citrate buffer (pH 6.0) or in EDTA buffer (pH 8.0) was effective in improving the efficiency of immunolabeling. However, ultrathin epoxy-embedded sections that were microwaved in alkaline solution (pH 10) were adequately labeled (5.2 +/- 0.34 particles per SG). Moreover, considerably improved efficiency of immunostaining was achieved by microwaving sections in alkaline solution (pH 10) with subsequent immunostaining at 60C (12.2 +/- 0.51 particles per SG). This method can also be applied to epoxy-embedded sections obtained from formalin-fixed, paraffin-embedded blocks of tissue and was even valid for an old epoxy-embedded block of tissue prepared 15 years previously.

Criteria for predicting the outcome of pheochromocytoma by the immunohistochemical and electron microscopic findings.

Morphological examination of 22 functionally active adrenal pheochromocytomas was carried out. The content of catecholamine granules in tumor cells and in the number of sustentacular cells tended to decrease in metastasizing tumors. Electron microscopy showed two types of sustentacular cells and the possibility of their apoptotic death.

Ultrastructural criteria of malignancy of adrenal medullary tumor.

Histological analysis and electron microscopy of 12 benign pheochromocytomas and 9 malignant pheochromoblastomas showed that there are no reliable histological differences between cells of benign and malignant tumor of the adrenal medulla. The ratio of ultrastructurally differentiated and undifferentiated cells in the tumor can reflect their maturity; the more ultrastructurally undifferentiated and less differentiated cells in the tumor, the higher is the malignant potential of this tumor.

Adrenal cortical adenoma with adrenalin-type neurosecretory granules clinically mimicking a pheochromocytoma.

Adrenal tumors often present with clinical features that are specific and unique to their endocrine metabolism. When these features are in conflict with the pathologic appearance of the tumor, there can be great consternation for both the pathologist and the surgeon. In the case reported herein, an adrenalectomy was performed for clinical features of pheochromocytoma that on gross and histologic examination had the pathologic features of an adrenal cortical adenoma. Electron microscopy subsequently revealed that the tumor cells contained adrenalin-type granules, explaining the clinical outcome. It is crucial for both the surgeon and the surgical pathologist to be aware of this possibility when the clinical and pathologic features of an adrenal tumor are not congruent.

Imaging of freeze-fractured cells with in situ fluorescence and time-of-flight secondary ion mass spectrometry.

Bioanalytical imaging techniques have been employed to investigate cellular composition at the single-cell and subcellular regimes. Four imaging modes have been performed sequentially in situ to demonstrate the utility of a more integrated approach to imaging cells. The combination of bright-field, scanning ion, and fluorescence microscopy complements TOF-SIMS imaging of native biomolecules. Bright-field microscopy provides a blurred visualization of cells in frozen-hydrated samples, while scanning ion imaging provides a morphological view of freeze-fractured cells after TOF-SIMS analysis is completed. With the use of selective fluorescent labels, fluorescence microscopy allows single mammalian cells to be located in the complex ice matrix of freeze-fractured samples, a task that has not been routine with either bright-field or TOF-SIMS. A fluorescent label, DiI (m/z 834), that does not interfere with the mass spectra of membrane phosphatidylcholine, has been chosen for fluorescence and TOF-SIMS imaging of membrane phospholipids. In this paper, in situ fluorescence microscopy allows the distinction of single cells from ice and other sample debris, previously not possible with bright-field or scanning ion imaging. Once cells are located, TOF-SIMS imaging reveals the localization of membrane lipids, even in the membrane of a single 15-microm rat pheochromocytoma cell. The utility of mapping lipids in the membranes of single cells using this integrated approach will provide more understanding of the functional role of specific lipids in functions of cellular membranes.

Identification of cellular sections with imaging mass spectrometry following freeze fracture.

Freeze-fracture techniques have been used to maintain chemical heterogeneity of frozen-hydrated mammalian cells for static TOF-SIMS imaging. The effects the fracture plane has on scanning electron microscopy and dynamic SIMS images of cells have been studied, but the implications this preparation method has on static SIMS have not been addressed to date. Interestingly, the chemical specificity and surface sensitivity of TOF-SIMS have allowed the identification of unique sections of rat pheochromocytoma cells exposed to the sample surface during freeze fracture. Using the extensive chemical information of the fractured surface, cellular sections have been determined using TOF-SIMS images of water, sodium, potassium, hydrocarbons, phosphocholine, and DiI, a fluorescent dye that remains in the outer leaflet of the cell membrane. Higher amounts of potassium have been imaged inside a cell versus the surrounding matrix in a cross-fractured cell. In other fractures exposing the cell membrane, phosphocholine and DiI have been imaged on the outer leaflet of the cell membrane, while phosphocholine alone has been imaged on the inner leaflet. In this paper, we discuss how imaging mass spectrometry isused to uniquely distinguish three possible sections of cells obtained during freeze fracture. The identification of these sections is important in choosing cells with a region of interest, like the cell membrane, exposed to the surface for a more thorough investigation with imaging static TOF-SIMS.

Ultrastructure of human pheochromocytoma cells cultured for long periods.

We conducted ultrastructural analysis of human pheochromocytoma (PC) cells maintained in primary culture for about 10 months. The cells were first isolated by the enzymatic treatment of a surgically resected tissue specimen obtained from a 37-year-old man with PC, a condition which is characterized by elevated blood levels of adrenaline and noradrenaline. It was found that noradrenaline production in the medium continued until the 90th day of culture (1330 pg/ml). The production level decreased to 20 pg/ml on the 180th day, and to 18 pg/ml on the 300th day. Examination under a transmission electron microscope (TEM) at 4 weeks of culture revealed electron-dense granules (about 200 nm in size and, presumably, rich in catecholamines), which were also observed in the tumor cells from the original PC tissue. Neurite-like processes grew at around 1 week of culture, and were still maintained at 6 months of culture. But, after 6 months of culture, the neurite-like processes contained a rosary-like elevated structure, which was suggestive of cell degeneration, as determined by a plasma polymerization replica method and observed with a scanning electron microscope. When cells were examined under the TEM, fewer electron-dense granules were observed in the cell bodies, with more numerous lipofuscin-like granules and filaments. Thus, electron-dense granules, which, presumably, contain catecholamines, were seen in a long-term culture of human PC cells. These granules decreased in number in parallel with the decrease in catecholamine levels in the culture.

Electron microscopic study to compare preclinical Cushing's syndrome with overt Cushing's syndrome.

BACKGROUND: No significant differences in gross and light- microscopic features have been reported between preclinical and overt Cushing's adenomas. In this study, the ultrastructural differences between the two syndromes was attempted to be clarified. METHODS: Two preclinical Cushing's syndrome adenomas and two overt Cushing's syndrome adenomas obtained from surgical extirpation were examined in an electron microscopic study. RESULTS: Light microscopically, the adenomas of both syndromes were composed predominantly of clear cells, with few compact cells. Ultrastructurally, the prominent differences were of development in each organelle: the preclinical Cushing's adenomas had undeveloped mitochondria, which were smaller in size and had sparse cristae, lysosomes and polysomes, whereas the overt Cushing's adenomas contained well-developed mitochondria which were larger in size and were filled with abundant cristae, smooth endoplasmic reticulum (SER), lysosomes and polysomes. CONCLUSIONS: Preclinical Cushing's syndrome adenomas were ultrastructurally characterized by a reduced number of cellular organelles such as mitochondria and SER, which are necessary to synthesize glucocorticoid hormones. However, examination of a greater number of adenomas will be required to be able to draw conclusions on the ultrastructural differences between the two syndromes.

[Expression of argyrophilic proteins from the nucleolar organizer regions as an index of maturity degree of benign and malignant adrenal tumors]. / Ekspressiia argirofil'nykh belkov oblastei iadryshkovogo organizatora kak pokazatel' stepeni zrelosti dobrokachestvennykh i zlokachestvennykh opukholei nadpochechnika.

Expression of APNOR was studied in 26 benign and malignant tumors of adrenal cortex and medulla. The histochemical method with silver nitrate was used. Expression of APNOR argyrophilic proteins in adrenocortical carcinomas was 4.59 times higher than in adrenocortical adenomas and 2.63 times higher in pheochromoblastomas than in pheochromocytomas. This index may be recommended as an additional method for differential diagnosis of benign and malignant adrenal tumors, particularly in difficult border line cases as well as determination of prognosis and metastatic potential of these tumors. Cytospecificity of APNOR expression in different types of adrenal cells was established.